Letters pubs.acs.org/acschemicalbiology
Orthogonal Control of Antibacterial Activity with Light Willem A. Velema,† Jan Pieter van der Berg,‡ Wiktor Szymanski,† Arnold J. M. Driessen,‡,§ and Ben L. Feringa*,†,§ †
Centre for Systems Chemistry, Stratingh Institute for Chemistry and §Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands ‡ Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, Nijenborgh 7, 9747 AG, Groningen, The Netherlands S Supporting Information *
ABSTRACT: Selection of a single bacterial strain out of a mixture of microorganisms is of crucial importance in healthcare and microbiology research. Novel approaches that can externally control bacterial selection are a valuable addition to the microbiology toolbox. In this proof-of-concept, two complementary antibiotics are protected with photocleavable groups that can be orthogonally addressed with different wavelengths of light. This allows for the light-triggered selection of a single bacterial strain out of a mixture of multiple strains, by choosing the right wavelength. Further improvement toward additional orthogonally addressable antibiotics might ultimately lead to a novel methodology for bacterial selection in complex populations.
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necessary to address the activity of multiple antimicrobial agents separately. Photocontrol over multiple events in parallel has recently been shown for functional surfaces,22 protein kinase activation,23 and photodeprotection of oligonucleotides.13 Remarkable reports of selective photochemical control24,25 include glutamate and GABA uncaging26,27 and photoinitiation and -inhibition for photolithography.28 Using various photocleavable protecting groups,29−31 with a significant difference in λmax, allows for the control of multiple processes in an orthogonal fashion. Numerous photocleavable protecting groups are available, such as o-nitrobenzyl groups, coumarins, and arylcarbonylmethyl groups.29−32 Here we describe a proof-of-concept for the orthogonal photocontrol over bacterial growth. First, the design of two antibiotics with orthogonally phototriggered activity is discussed, regarding their photochemical and antimicrobial properties in response to irradiation. Subsequently, analysis of the photochemistry of the two caged antibiotics is described. The antibacterial properties of the designed compounds were determined before and after photoactivation, for Escherichia coli and Staphylococcus aureus. Finally, the orthogonal release of antimicrobial substances is used for bacterial selection. This system can be triggered by two different wavelengths, where the absence or presence of light at these wavelengths induces selective growth of E. coli and S. aureus. Within this proof-of-
icroorganisms are able to coexist in mixed populations, which can cause complex bacterial infections, i.e., infections with two or more unrelated strains, and what can also be a source of sample infections in microbiology practice.1 Specifically addressing a single bacterial strain2,3 from a mixture of multiple strains is a fascinating scientific challenge and a necessity in healthcare for diagnostics and screening and provides an important tool in microbiology research.4 Of special interest are methods that attain bacterial selection using an external trigger, with minimal perturbation to the studied microbial population. Furthermore, it would be advantageous if this trigger could be delivered with high spatiotemporal precision, as it not only allows for externally controlled bacterial selection but might be exploited for precise bacterial patterning as well.5,6 The use of light as an external trigger to control biological processes, such as cellular growth, is an interesting option, since the delivery of light can be precisely controlled in space and time. Additionally, light is relatively noninvasive and bioorthogonal and does not cause sample contamination.7−12 Furthermore, both the qualitative (wavelength) and quantitative (amplitude) properties of light can be precisely controlled, allowing independent and regulated activation of several species.13 Significant progress has been made in achieving optical control over biological processes14 using photoisomerisable moieties.8,15,16 Recent examples include the remote control of protein channel function,17 nociception regulators,18 enzyme inhibitors,19,20 mast cell activation inhibitors,21 and antibiotics.5 However, in all of these cases only a single process was targeted. To develop a system for bacterial selection, it is © 2014 American Chemical Society
Received: April 28, 2014 Accepted: July 23, 2014 Published: July 23, 2014 1969
dx.doi.org/10.1021/cb500313f | ACS Chem. Biol. 2014, 9, 1969−1974
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Figure 1. Schematic representation of the required elements for light-triggered bacterial selection. Two complementary antibiotics that can be orthogonally activated with different wavelengths of light are combined with two different bacteria. Activation of antibiotic 1 causes a growth inhibition of bacteria 1 and results in growth of bacteria 2. When antibiotic 2 is activated, the growth of bacteria 2 is inhibited and growth of bacteria 1 is realized. When none of the antibiotics is activated, bacteria 1 and bacteria 2 will grow. Activation of both antibiotics leads to death of both bacteria.
group would result in significant loss of activity, which would be regained after photolysis. The carboxylate group of compound 2 interacts with the binding site of transpeptidase,34 and its modification into a photolyzable ester reduces the antimicrobial activity, which can be restored after photodeprotection. Derivatives of 7-dialkylaminocoumarin and 7-alkoxycoumarin were used as photocleavable groups (Figure 2a). These compounds show absorption bands with a λmax of ∼380 nm and ∼310 nm, respectively, which allows for orthogonal photoactivation. Furthermore, the amino and hydroxyl groups can be alkylated with carboxylic acid-bearing compounds, which renders these molecules water-soluble. The two designed photoactivatable antibiotics are shown in Figure 2a. The fluoroquinolone analogue was named FQNC (FluoroQuinolone-N-Coumarin) (Figure 2a) and was protected with a disubstituted 7-aminocoumarin group, which allows for photoactivation at 380 nm (vide inf ra). The photoactivatable benzylpenicillin (Figure 2a) bears a substituted 7-hydroxycoumarin moiety that could be released by irradiation with 312 nm light (vide inf ra). This compound was named BPOC (BenzylPenicillin-O-Coumarin). UV−vis spectroscopy was used to study the photochemical properties of the photoactivatable antibiotics. An absorption band for FQNC with a λmax of 381 nm was observed (Figure 2b). Irradiation with white light resulted in a decrease of this absorption band, suggesting that the photolyzable coumarin group was cleaved and 1 was liberated. BPOC has an absorption maximum at 322 nm (Figure 2b). When this sample was illuminated with 312 nm light, a decrease in absorption was observed. This decrease is caused by the photodeprotection of BPOC, which results in the release of benzylpenicillin 2. To examine the orthogonality of this photocleavage process, the rate of photodeprotection was studied for FQNC and BPOC with 312 nm and white light
concept, we report on the selection of either one bacterial strain out of a mixture of two. However, rapid advances in the field of photochemistry and the vastly growing number of orthogonally-addressable photocleavable groups will likely extend this method to be applicable for bacterial selection out of more complex mixtures of microorganisms.
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RESULTS AND DISCUSSION When designing a system that allows for orthogonally controlling bacterial growth, several factors need to be taken into account. First, two antibiotics should be chosen that are complementary in their antibacterial spectrum, i.e., when using a mixture of two bacteria, the first antibiotic inhibits the growth of only one bacterium but does not affect the growth of the other and the second antibiotic does the opposite. A second consideration is the ease by which the antibiotics can be caged with a photolyzable group. Carboxylic acid moieties present in the antimicrobial agents are especially suitable for this purpose. Furthermore, the protected antibiotics must show significantly lower activity when compared to the native ones. Another prerequisite is sufficient water solubility of the designed compounds. When all of these design elements are combined, a system can be assembled as schematically depicted in Figure 1. On the basis of the above-mentioned considerations, a fluoroquinolone (1) and benzylpenicillin (2) were selected as antibiotics (Figure 2a). The two bacteria chosen for this system were E. coli and S. aureus. E. coli is susceptible to the fluoroquinolone (1) but is not affected by benzylpenicillin (2), whereas S. aureus is sensitive for 2, but is not affected by the fluoroquinolone (vide inf ra). Additionally, both antibiotics bear a carboxylic acid functionality, which can be protected with a photolyzable group. The carboxylic group of fluoroquinolones is essential for binding to DNA bases,33 and caging of this 1970
dx.doi.org/10.1021/cb500313f | ACS Chem. Biol. 2014, 9, 1969−1974
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Figure 2. Molecular structure of designed photoactivatable antibiotics and products liberated after photocleavage and change in UV−vis absorption spectra. (a) FQNC consists of a fluoroquinolone that was protected at its carboxylic acid group with a derivative of 7-aminocoumarin. The coumarin moiety can be cleaved from FQNC by illumination with 380 nm light, liberating the free carboxylic acid functionality of the fluoroquinolone (1). The molecular structure of BPOC comprises a benzylpenicillin conjugated with a derivative of 7-hydroxycoumarin at its carboxylic moiety. Exposing BPOC to 312 nm light releases benzylpenicillin. (b) The UV−vis spectrum of FQNC shows an absorption maximum at 381 nm as well as significant absorbance between 300 and 350 nm. BPOC has one absorption band with a maximum around 320 nm. (c, d) The photocleavage of FQNC (25 μM in water) and BPOC (18 μM in water) was measured over time by observing the decrease in absorbance at λmax (381 and 322 nm, respectively) using UV−vis spectroscopy.
8.9 × 10−3 (at 376 nm), respectively. Furthermore, the compounds were stable for at least 4 h when dissolved in water (Supplementary Figure S5−S6). FQNC and BPOC are the carboxyl-protected analogues of 1 and 2, respectively. The bacteria used for this study were E. coli CS1562 (tolC6:tn10)35 and S. aureus RN4220.36 Compound 1 has antibacterial activity against E. coli with a minimal inhibitory concentration (MIC) (lowest concentration that inhibits bacterial growth) of 68 μM (Figure 3a and Supplementary Figure S7) and has significantly lower activity against S. aureus with a MIC of >1700 μM (Figure 3a and Supplementary Figure S7). In contrast, 2 has high activity against S. aureus with a MIC of 350 nM (Figure 3a and Supplementary Figure S8) and dramatically less activity against E. coli with a MIC of 288 μM (Figure 3a and Supplementary Figure S8). Figure 3b shows bacterial growth curves of E. coli when incubated with 76 μM FQNC, before and after exposure to white light (see Supplementary Figure S9 for MIC determination of FQNC). Before exposure to white light, normal bacterial growth was observed, comparable to the control without any antibiotic present. After irradiating the samples for 5 min with white light,
irradiation (Figure 2c and d), by observing the decrease in absorption at λmax. Figure 2c shows that FQNC could be deprotected by irradiation with white light (150 W, 1 cm distance, see Supplementary Figure S1 for spectrum). Illuminating a sample with 312 nm light (8 W, 2 cm distance) also resulted in complete deprotection. This might be caused by a strong absorption of the quinolone moiety at these wavelengths, which may result in energy transfer, preventing selective deprotection of FQNC by white light only. However, FQNC and BPOC have a large difference in antibacterial activity, and exploiting these properties results in orthogonal growth of E. coli and S. aureus (vide inf ra). The photodeprotection rate was also studied for BPOC (Figure 2d). When BPOC was exposed to white light, only a small decrease in absorption was observed, whereas a much more dramatic decrease in absorption was achieved using 312 nm light irradiation. This enables selective activation of BPOC with UV light. To further confirm the liberation of 1 from FQNC and 2 from BPOC after exposure to light, 1H NMR spectroscopy and HR-MS were used to characterize the deprotected antibiotics (Supplementary Figure S2−S4). The quantum yields of FQNC and BPOC were determined to be 7.2 × 10−4 (at 323 nm) and 1971
dx.doi.org/10.1021/cb500313f | ACS Chem. Biol. 2014, 9, 1969−1974
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Figure 3. MIC values and bacterial growth curves of E. coli and S. aureus incubated with FQNC and BPOC before and after photodeprotection. (a) MIC values of compounds 1 and 2. (b) When E. coli was incubated with 76 μM FQNC, it exhibited a normal growth pattern comparable to that of the control without FQNC. When the samples were exposed to white light prior to incubation, E. coli growth was inhibited. (c) The growth of S. aureus was significantly inhibited when incubated with 880 nM BPOC that was irradiated at 312 nm, as compared to S. aureus that was incubated with 880 nM BPOC that had not been exposed to light. Error bars show standard deviations calculated from measurements in triplicate.
bacterial growth was inhibited. This indicates that 1 was liberated after white light exposure, inhibiting bacterial growth. Next, a similar experiment was conducted for BPOC. Samples of BPOC (880 nM) that were exposed to 312 nm for 2 min, prior to incubation with S. aureus, showed minimal bacterial growth (Figure 3c). When S. aureus was incubated with samples with the same concentration of BPOC but without exposure to light, significantly more bacterial growth was observed. The MIC of BPOC against S. aureus was 3.5 μM without exposure (Supplementary Figure S9) and 880 nM with exposure to 312 nm light. These experiments indicate that the antibiotic activity of the two protected compounds FQNC and BPOC can be induced at two distinct different wavelengths. Furthermore, the liberated antibiotics show a large difference in spectrum of activity, as was expected, and therefore inhibit bacterial growth of different bacterial strains. This is an important prerequisite for realizing our system for bacterial selection. By combining these properties, FQNC and BPOC can be employed for light-triggered bacterial selection. To realize this, agar plates were prepared that contained 48 μM FQNC and 350 nM BPOC. Next, the agar plates were kept in the dark or exposed to 312 nm light (10 s), white light (5 min), or both 312 nm and white light. Subsequently, the plates were divided in three equal parts (Figure 4a). One part was inoculated with E. coli (‘E. coli’ sector), another part with S. aureus (‘S. aureus’ sector), and the third part was inoculated with both bacteria (“both” sector). After incubation overnight, (the lack of) bacterial growth could be observed (Figure 4a). The plate (P1) that was not exposed to any light showed bacterial growth in all three sectors, indicating that E. coli, as well as S. aureus, were growing. Upon closer examination, a distinct difference in morphology between the colonies of E. coli and S. aureus could be observed (Figure 4b). At the “both” sector both morphologies were present (Figure 4b). The second plate (P2) that was exposed to 312 nm light showed no growth at the S. aureus sector and normal growth at the other
Figure 4. Light-triggered bacterial selection. (a) Agar plates containing FQNC (48 μM) and BPOC (350 nM) were kept in the dark or exposed to 312 nm light, white light, or a combination of 312 nm light and white light. Subsequently, E. coli and S. aureus were inoculated at different sectors on the plate. Overnight incubation resulted in bacterial growth in selected sectors on the agar plates. (b) A close-up of the colonies formed by E. coli and S. aureus. There was a distinct difference in morphology of colonies of the two bacterial strains. When the two bacteria were grown together with and without the photoactivatable antibiotics, the two different morphologies were distinguishable.
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were determined by calculating the slope of the exponential growth phase. MIC values were calculated by plotting the growth rates against the concentrations of the used antibiotics.
two sectors, which implies that only E. coli was growing, which was also evident from the colony morphology. The third plate (P3) was exposed to white light and did not show any growth at the E. coli sector, whereas normal growth was observed in the other two sectors. This suggests that only S. aureus was growing, also evidenced by colony morphology. The last plate (P4) was exposed to 312 nm and white light, and after incubating overnight, almost no bacterial growth was observed in any of the sectors, indicating that E. coli as well as S. aureus growth was inhibited. These observations prove that FQNC and BPOC can be employed to select each of the bacterial strains out of a mixture of two strains. This proof-of-concept shows that light can be used as an external trigger to control bacterial selection. This concept holds promise to be expanded to a larger number of orthogonally-activated antibiotics, which will allow for bacterial selection from more complex mixtures of microorganisms, using light. Such future studies will be supported by the rapid advancement in the design of orthogonally addressable photocleavable groups. Complex mixtures of various bacterial strains might be analyzed and cell populations selected, by simply exposing the system to the right wavelengths of irradiation. The methodology presented here represents a valuable addition to the toolbox for bacterial selection and might also be particularly useful for high-precision patterning of various microorganisms in one system.
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ASSOCIATED CONTENT
S Supporting Information *
Additional figures and synthetic procedures. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by The Netherlands Organization for Scientific Research (NWO-CW), The Royal Netherlands Academy of Arts and Sciences Science (KNAW) and the European Research Council (ERC) advanced grant 227897 to B.L.F., and the Ministry of Education, Culture and Science (Gravity programme no. 024.001.035).
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METHODS
REFERENCES
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Synthesis of FQNC and BPOC. The synthesis of FQNC and BPOC is described in the Supporting Information. Uncaging Experiments. Irradiation experiments were performed with a Spectroline ENB-280C/FE UV lamp (312 nm) and Thor Laboratories OSL1-EC Fiber Illuminator (white light) (see Supplementary Figure S1 for lamp emission spectrum). Quantum Yield Determination. Quantum yields were determined by irradiating 2.5 μM solutions of BPOC and FQNC with 323 and 376 nm light, respectively, with a JASCO FP-6200 spectrofluorometer. After each irradiation period, UV−vis absorbance was measured. The percentage of photolysis was estimated by the decrease in absorbance at λmax. Quantum yields (Q) of photolysis were calculated using the following equation: Q = −log(Ct/C0)/Iσt. Ct and C0 are the concentrations of BPOC or FQNC at time t and time 0, respectively. σ is the decadic extinction coefficient in cm2·mol−1, t is the irradiation time in seconds, and I is the light intensity in einsteins· cm−2·s−1 that was determined using ferrioxalate actinometry.37 Bacterial Strains and Growth Conditions. The bacterial strains used were E. coli CS1562 (tolC6:tn10)35 and S. aureus RN4220.36 Both strains were grown in LB medium (5 g/L yeast extract; 10 g/L tryptone; 0.5 g/L NaCl) supplemented with the required antibiotic at 37 °C. Solid Medium. An LB agar plate containing FQNC and BPOC was not irradiated with light or irradiated with UV light for 10 s and/or visible light for 5 min. The plate was then streaked with approximately 104 CFUs of E. coli CS1562 and S. aureus RN4220 and incubated overnight at 37 °C. Antibacterial Activity and Bacterial Growth Curves. Overnight cultures of E. coli CS1562 and S. aureus RN4220 were diluted to an OD600 of 0.1, and 100 μL of this cell suspension was added to 100 μL of medium containing antibiotics at the given concentration. To determine the antibacterial activity after light exposure, the solutions were first irradiated at 312 nm for 2 min or white light for 5 min prior to adding the cell suspension. Cells were grown in a microtiter plate at 37 °C, and cell density (OD at 600 nm) was measured every 10 min for 12 h, with a 10 s shaking step before each measurement, in a microplate reader (SynergyMX, BioTek). Graphs were backgroundcorrected by subtracting the OD600 at time 0. Before calculating the growth rate, graphs were plotted on a logarithmic scale. Growth rates 1973
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